Riboswitches are genetic control elements located within the 5' untranslated regions of messenger RNAs (mRNAs) that undergo metabolite-dependent structural rearrangements to regulate mRNA transcription, splicing, translation, or stability in response to the presence and concentration of specific metabolites. The ubiquity of riboswitch-mediated transcriptional control in bacteria and the specificity with which riboswitches control bacterial gene expression are fueling efforts to develop next-generation antibiotics that target bacterial riboswitches. In addition, riboswitches are quickly becoming powerful tools in the field of synthetic biology, where they can be engineered to artificially control gene expression. Fully exploiting riboswitches for these applications, however, requires a detailed understanding of the mechanism of riboswitch-mediated transcriptional control. Although single-molecule (sm) biophysical methods, including sm fluorescence microscopy and sm force microscopy, have established themselves as powerful tools for studying metabolite- dependent structural rearrangements of riboswitches and transcription by RNA polymerases (RNAPs), the mechanistic information available from these sm methods remains limited by technical obstacles such as: (i) difficulties in fluorophore labeling of biomolecules; (ii) the application of invasive artificial forces; (iii) limited time resolution; and (iv) limited total observation time. The higly multi-disciplinary effort described here will expand upon recent development of a carbon nanotube-based sm field effect transistor (smFET) as a new, label-free, non-invasive, high-time-resolution, extended-observation-time, sm method for in vitro studies of biomolecular binding kinetics and structural dynamics. This smFET-based experimental system will be further developed to overcome many of the limitations of established sm methods, enabling the Bacillus subtilis pbuE adenine-responsive riboswitch and the corresponding B. subtilis RNAP to be used as a model system for studying the mechanisms of metabolite- dependent riboswitch structural rearrangement (Aim 1), transcription (Aim 2), and real-time riboswitch- mediated transcriptional control (Aim 3) at unprecedented time resolutions and throughputs. These studies will enable characterization of some of the most poorly defined aspects of the mechanism of riboswitch-mediated transcriptional regulation and will provide the tools and knowledge necessary to drive the development of new antibiotic drugs that target bacterial riboswitches and the design of new riboswitches that can be used to regulate synthetic gene networks.